Hepatitis C virus (HCV) often causes a prolonged and persistent infection. Association between hepatocellular carcinoma (HCC) and HCV infection has been noted. Immune evasion and quasi-species nature are prominent features of HCV. Understanding the molecular basis of viral pathogenesis is a major challenge to gain insight into HCV associated disease progression. The pathogenesis of liver damage is likely to be related to both viral and immune mediated factors. Recent experimental evidence using HCV cloned genomic regions suggests that the NS5A protein has many intriguing properties. These include the presence of an interferon sensitivity determining region (ISDR), direct repression of PKR, a regulatory role on important cellular promoters, and promotion of cellular transformation. Together these observations provide a compelling reason to focus and design studies to further our understanding of the molecular mechanism and cellular targets of HCV NS5A protein mediated biological functions. Available information suggests that during persistent infection NS5A protein may play a critical role in concert with cellular factors for virus mediated pathogenesis. This research proposal is designed to (1) Determine the role of HCV NS5A protein on cell cycle regulatory genes and map the transregulatory domain; (2) Investigation the interaction of HCV NS5A protein with cellular target protein(s); (3) Determine the role of HCV NS5A protein in cytokine mediated apoptosis; (4) Determine the transforming potential of HCV NS5A protein in primary human hepatocytes; and (5) Determine whether HCV NS5A protein expression promotes liver pathogenesis in transgenic mice. Results from this study will lead to a better understanding of the virus mediated pathogenesis. The long term goal of this study is to design effective strategies for the intervention of HCV mediated disease in humans.